Nijmegen breakage syndrome (NBS) is characterized by hyper-sensitivity to ionizing radiation, high frequency of chromosomal aberration, and high incidence of malignancy. NBS1, the underlying protein for NBS, form a complex with MRE11/RAD50 (M/R), and plays a crucial role in DNA repair processes by regulating the M/R activities and localization. NBS1 complex are also involved in DNA damage signaling, such as cell cycle checkpoint activation and apoptosis induction, through regulation of ATM activation and localization. Using hyper-recombinogenic DT40 cells, we found that apoptosis induction after irradiation was significantly suppressed in Nbs1 knockout cells compared to wild type cells. Because p53 expression was lost in DT40 cells, our observation suggests that Nbs1 might be involved in apoptosis induction pathway that is independent of p53. Results from further analysis of p53 independent apoptosis induction pathway after irradiation, which is regulated by NBS1, will be discussed.

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In this study, we analyzed the morphological changes in the cells that were irradiated with UV or ionizing radiation (IR) to clarify the molecular mechanism of cell death which is induced by DNA damages. The RIC (Radiation Induced Curly tailed malformation) mutants are isolated by ENU mutagenesis screening in the Medaka. We have reported that RIC1 mutant has the defect in the mechanism that repair the double-strand breaks of DNA stands induced by IR in embryos. We established cultured cell lines derived from wild-type (CAB strain) and ric (RIC1 and RIC2) and the time-laps observation of morphological change after irradiation of IR, UVA, and UVC was performed. When we irradiated the cells with 20Gy of IR, the giant cells and cells with many vacuoles were observed and such cells were observed more frequently in RIC1 cells than in wild type cells. When CAB and RIC1-derived cells were irradiated with UVC (20J/m²), the fibroblast-like cells became rounded and detached from the bottom of the culture dish, then fragmented into small pieces and finally died. RIC1 cells were more sensitive to UVC irradiation than CAB cells. When CAB cells were irradiated with UVA (50kJ/m²), there was little effect. UVA-irradiated RIC1 cells became rounded and detached from the bottom. After UVA irradiation, RIC2 cells still attached to the bottom of the culture dish and maintained their pseudopodia and their fibroblast-like figure, but their nuclei were clarified, just same as in fixed cells.

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Mouse lymph node cell (M10) is highly sensitive to X-ray irradiation. This cell is also sensitive against UV-ray irradiation. Growth of this cell depended on the UV-rays (310 nm, band B) irradiation, and the cell growth was suppressed in proportion to the UV irradiation time. UV irradiation doses (10, 20, 30 and 60sec) showed the induction of cell apoptosis and necrosis. Cell sorting analysis distinguished the difference of the apoptosis and the necrosis of M10 cells. Apoptotic cells increased in proportion to UV-irradiation. We also analyzed the gene expression in the UV-irradiated cells using DNA array technology. Dynamic changes of 597 genes after the UV-B (310 nm) irradiation of three doses (10, 20 and 30sec) were investigated on DNA array membranes. Radioactivities obtained from imaging analyzer were analyzed using software of ArrayGauge. The expression of mRNA was further analyzed by EX-ARRAY program. Time course of the gene expression was investigated at 2h and 5h after the irradiation. The expression of almost genes reduced in proportion to the doses. The comparison of the gene expression with the parental cells (L5178Y) demonstrated the remarkable activation of homeobox gene (Hox-2.4) and serine (or cysteine) proteinase inhibitor, clade B member 6 (Serpinb6).

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Most hematopoietic cells, such as thymocytes and lymphocytes, are known to be hypersensitive to radiation and exhibit interphase cell death with a typical morphological characteristic of apoptosis. In order to identify the signaling regulators associated with UV-induced apoptosis, we analyzed cellular proteins in Jurkat cells responding to UV irradiation using two dimensional gel electrophoresis (2DE).
Jurkat cells were irradiated with 20 J/m² of UV, incubated for various time intervals, and then treated with digitonin for preparing their cytosolic extracts. Comparison of the high resolution 2DE protein patterns of the extracts from irradiated and un-irradiated cells showed differences in many spots including protein modifications. Intensive protein spots appeared in UV-irradiated cells were analyzed after tryptic digestion by peptide mass fingerprinting using a MALDI-TOF/TOF mass spectrometry. Interestingly, one of 12 proteins identified, acidic ribosomal protein P2 (P2) which is known to be mainly located within the 60s ribosomal subunit, was detected in three different pI spots, indicating the presence of two phosphorylation residues in cytoplasmic P2. Since only the phophorylated P2 was found in un-irradiated Jurkat cells and the amount of unphophorylated forms of P2 increased with increasing time of incubation after UV irradiation, the dephosphorylation of P2 may be associated with the activation of signaling pathways leading to apoptosis in UV-irradiated Jurkat cells.

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We previously reported that p42/SETß is a substrate for caspase-7 in irradiated MOLT-4 cells, and that treating the cells with sodium orthovanadate (vanadate, Na₃VO₄) inhibits p42/SETß's caspase-mediated cleavage. Here, we initially found that the inhibitory effect of vanadate was due to the suppression of caspase activation but not of caspase activity. Further investigations revealed that vanadate suppressed upstream of apoptotic events, such as the loss of mitochondrial membrane potential, the conformational change of Bax, and p53 transactivation, although the accumulation, total phosphorylation, and phosphorylation of six individual sites of p53 were not affected. Importantly, vanadate suppressed p53-dependent apoptosis, but not p53-independent apoptosis. Finally, gel-shift and chromatin immunoprecipitation (ChIP) assays conclusively demonstrated that vanadate inhibits the DNA-binding activity of p53. Vanadate is conventionally used as an inhibitor of protein tyrosine phosphatases (PTPs); however, we recommend that the influence of vanadate not only on PTPs but also on p53 be considered before using it.

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Since UV generally causes a rapid cell death and ionizing radiation induces a delayed form of apoptosis, the activation of signal transduction by radiation would be different between γ-rays and UV. In order to identify the signaling regulators associated with apoptotic cell death, we analyzed cellular proteins responding to γ or UV irradiation using two dimensional gel electrophoresis (2DE). In this study, Jurkat cells were irradiated with 15 Gy of γ-rays or 20 J/m² of UV, both of which show a similar effect on cell killing, incubated for 48 h or 6 h respectively, and treated with digitonin for preparing their cytosolic extracts. Comparison of the silver stained 2DE protein patterns of the extracts from irradiated and un-irradiated cells showed differences in many spots. These protein spots were classified into three groups proteins; specifically responding to either γ-rays (A) or UV (B), and proteins responding to both radiations (C). Then the protein spots, belonging to the group (C), were digested by trypsin and analyzed by a MALDI-TOF/TOF mass spectrometry. Acidic ribosomal protein P2, Rho GDP dissociation inhibitor β, and initiation factor 5A were identified as a protein responding to both irradiations by means of peptide mass fingerprinting analysis.

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Recently, we reported additive or synergistical enhancement of X-irradiation-induced apoptosis by TRAIL (TNF-related apoptosis-inducing ligand) in solid tumor cell lines. However, the mechanism of signal transduction for apoptosis in the cell treated with the combination of TRAIL + X-irradiation is still unknown. In this experiment, to clarify the mechanism of signal transduction in the combination of TRAIL / X-irradiation-induced apoptosis in lung cancer cell line A549, the amount of apoptosis was measured by flow cytometer after the treatment of combination of X-irradiation + TRAIL, X-irradiation alone and TRAIL alone. The apoptotic cell death exposed to TRAIL + X-irradiation synergistically increased compared with that of TRAIL alone or of X-irradiation alone. In cells exposed to TRAIL alone, western blotting showed that the fragmentation of XIAP was observed and the expression of survivin and Bcl-X_L significantly increased. In the case of X-irradiation alone, no fragmentation of XIAP was induced and, downregulation of survivin and upregulation of Bcl-X_L occurred. In contrast, the increase in the fragmentation of XIAP and the down regulation of survivin and Bcl-X_L were observed in the cells treated with the combination of TRAIL + X-irradiation. These data suggest that the strong downregulation of antiapoptotic proteins, i.e., survivin and Bcl-X_L, induced by the combination treatment was responsible for synergestical enhancement of radiation-induced apoptosis in A549 cells.

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We have reported that remarkable apoptosis was induced by gamma ray-irradiation in peritoneal resident macrophages (PRM) of C3H mice, but not the other strains of mice and that superoxide played the major role in the apoptosis. In the present study, we focused on antiapoptotic proteins, particularly Bcl-2 family proteins because proteasome inhibitors, MG132 or lactacystin, significantly suppressed irradiation-induced apoptosis in C3H mouse PRM. Western blot analysis revealed that irradiation did not affect the amounts of Bcl-2, Bax, Bcl-X_L, A1, cIAP-1 but markedly decreased the amount of Mcl-1 protein from 2hr after irradiation. Mcl-1 protein was decreased dose-dependently, and the decrease in Mcl-1 protein was well correlated with the increase in apoptosis in C3H mouse PRM. Mcl-1 protein level in B6 mouse PRM resistant with respect to irradiation-induced apoptosis was not altered with irradiation. On the other hand, Mcl-1 mRNA measured by real time PCR markedly increased in C3H mouse PRM with irradiation. Therefore, irradiation-induced depletion in Mcl-1 protein of C3H mouse PRM was thought to be due to the translational inhibition or enhanced proteasomal degradation of the protein. The experiment with antisense in order to demonstrate whether apoptosis is induced in PRM by depletion of Mcl-1 is in progress.

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p53 is a tumor suppressor protein. It is shown that over 50% of all human tumors carry inactivating mutations in the p53 gene. In contrast to human leukemia MOLT-4 cells (p53 wild-type) which develop apoptosis after X-irradiation, mouse leukemic M10 cells (p53 mutant) undergo necrosis (Nakano and Shinohara, 1994). In addition, mild hyperthermia induces apoptosis in M10 cells (Nakano et al. 1997). In the present study M10 cells were transfected with wild-type p53 cDNA vector using DMRIE-C reagent. The stable transfectants were cloned by the colony formation in the presence of puromycin These transfectants were examined by PCR analysis and Western blot analysis. The loss of cell viability was determined by the dye exclusion test. The fraction of the transfectants stained with erythrosine B was low in unirradiated state, and was increased dramatically within 24h of incubation after 10 Gy-irradiation, while only a slight increase was observed for M10 cells. The increase of dead cells was dependent on X-ray doses. These results suggest that transfection of wild p53 to mutant p53-tumor cells is effective for radiosensitization.

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The ric (Radiation Induced Curly tailed malformation) mutation is isolated by ENU mutagenesis screening in the Medaka. We reported that the ric1 phenotype is associated with defective rapid repair of DNA double-strand breaks induced by ionizing radiation (IR) in embryos (Aizawa et al 2004). We examined radiation effects on testis and intesitine histologically. At 24 hours after 2.5Gy γ-irradiation, a lot of apoptosis in spermatogonial stem cells (SSCs) were detected in the wild-type CAB strain, but in the ric1 testis, few pycnotic SSCs and spermatocytes were detected and filled with differentiating spermatogonia, and almost all spermatocyte disappeared. At 3days after irradiation, there was no pycnotic SSC in CAB but many were observed in ric1. Within 5days after irradiation almost all SSCs disappeared in ric1 and within 30days after irradiation they became sterile. These results show that the different pathways for DNA repair and apoptosis induction are employed during spermatogenesis and the ric1 gene product has an important role in triggering the early apoptosis in the SSCs and differentiating spermatogonia. The intestine of ric1 showed similar radiosensitivity to that of CAB, which means the ric1 is not involved intestinal stem cells. The ric1 locus was mapped in LG9, and about 400kb genomic region containing ric1 gene was determined. Now, we are examining the radiation effects on primordial germ cells (PGCs) in ric1 using olvas-ric1 strain obtained by the crossing of ric1 with olvas-GFP with germ cell specific expression of GFP.

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We thought to perform a partial hepatectomy (HP) after irradiation in order to induce cell proliferation and thereby increase the detection of mutations in mice. So we needed data about the dynamics of cell proliferation after HP in irradiated and non-irradiated mice.C57BL mice were irradiated with 5Gy of gamma rays before HP. The remnant livers of irradiated and non-irradiated mice were excised after pulse labeling for 1 hr with BrdU at 20, 24, 28, 32, 36, 40, 44, 48, 60, 72 hr after HP. The BrdU labeling Index (BLI) was calculated as the percentage of S-phase cells per liver cells. In the non-irradiated group, cell proliferation began about 32 hr after HP with the BLI indicating a peak at 36 hr. The BLI had decreased by 48 hr and then increased again at 60 hr, with a smaller peak than that observed at 36 hr. At 72 hr, the BLI had returned to background level. In the irradiated group, BLI dynamics were similar, but the levels of BLI at 36 hr and 60 hr after HP were significantly lower than the non-irradiated group.We had prediction that the timing of cell proliferation might be late or S-phase cells might accumulate in the irradiated group. However, the prediction is not supported by the data from this study. It appears that after irradiation, p53 products can rapidly distinguish between liver cells that are seriously damaged and those that are not. So the number of regenerating cells had already decreased before HP. According to Inoue et al., p53 products increase ahead of BLI after HP. Therefore, the increase in p53 products after HP could work in a similar fashion.

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Responses of two lines of feline T lymphocytes, FeT-J and FL-4, to ionizing radiation were examined in the present study. FL-4 cells, but not FeT-J, were persistently infected with feline immunodeficiency virus. Cells were irradiated with ⁶⁰Co-γ rays at 2 Gy/min. Surviving fractions were evaluated with clonogenic assay. Apoptosis was detected using TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Changes in nuclear morphology of viable cells were evaluated by double staining with Hoechst 33342 and ethidium bromide. Mean surviving fractions of FL-4 cells were slightly higher than FeT-J for each radiation dose given. However, dose required to reduce to 63 % of surviving fraction was 1.9 Gy in both cell lines. TUNEL assay, however, revealed the maximum frequency of apoptosis in FL-4 cells (<20 %) was lower than in FeT-J cells (>40 %). The time of frequency peak of TUNEL-;positive cells in FL-4 was shorter than that of FeT-J. FL-4 cells reached peak level within 24 hours after irradiation, but FeT-J cells needed later than 48 hours after irradiation. Exposure of FL-4 cells to γ rays resulted in the giant- and the multi- nucleus formation. Nuclear swelling occurred to less extent in FeT-J than FL-4 cells. In conclusion, we found the difference in cellular responses to radiation in two lines of feline T lymphocytes. Modes of cell death of FL-4 cells were non-apoptotic and more research would discover new mechanism associated with apoptosis.

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Survivin, a member of IAP ( inhibitor of apoptosis proteins ), is highly expressed in tumor cells. This protein is reported to be phosphorylated at threonine 34 (T34) by CDC2 and to suppress apoptosis by inhibiting caspases. Recently, the residue of aspartic acid 53 (D53) was also demonstrated to be important for the inhibition of apoptotic signaling. In this study, we constructed a replication-deficient adenovirus encoding survivin with point mutations (T34A and D53A) and examined their effects on radiation-induced apoptosis. The cDNAs consisting of GFP and wild type (WT) survivin or survivin mutants (T34A and D53A) were inserted in pAd/CMV/V5-DEST vector. Virus was produced in 293A cells and the crude viral supernatants were used to transduce human lung carcinoma A549 cells. The expression of GFP proteins was detected in all of the cells after transfection of GFP viral vector as confirmed by fluorescence microscopy. After irradiation, the apoptosis induction was evaluated by microscopic observation. Overexpression of survivin-T34A enhanced the radiation-induced apoptosis in comparison with that of wild type survivin. The result suggested that the point mutations at T34 abolished the anti-apoptotic activity of survivin and enhanced the induction of apoptosis in X-irradiated A549 cells. Examination of the effects of survivin-D53A on radiation-induced apoptosis was under progress.

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Reproductive processes are particularly radiosensitive in plant development, which was clearly illustrated in reduction of seed formation in native coniferous plants around Chernobyl after the nuclear accident. For the purpose to investigate the effects of ionizing radiation on embryonic formation in coniferous plants, we used an embryo-derived embryogenic cell culture of a Japanese native coniferous plant, Japanese cedar (Cryptomeria japonica). The cells were so radiosensitive that most of the cells died by acute X-ray irradiation of 0.5 Gy. The cell death was characteristic of nuclear DNA fragmentation, which is typically observed in apoptosis in mammalian cells. The apoptosis-like cell did not develop in vegetative tissues, i.e. meristematic cells, after X-ray irradiation, and considered to be peculiar to embryogenic cells. Development of cell death in the embryogenic cells was suppressed by inhibitors against MAPkinases, which was activated two hours after X ray irradiation of the cells. The results indicate that the embryogenic cells develop apoptosis-like programmed cell death, which should be mediated by signal transduction through MAPkinases. The abortion of embryogenic cells may work to prevent transmission of radiation-induced genetic damages to the descendants.

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§Introduction§ Biological effects by low dose rate irradiation is known to be different from those by high dose rate irradiation. Specific cellular (NIH/PG13Luc cells) responses and gene expression pattern for low dose rate irradiations have been detected by us. In present study, we observed focus formation of γH2AX and p53BP1 in murine NIH/PG13Luc cells.
§Materials and Methods§High dose rate irradiations (0.5 Gy/min) were performed by soft-X ray for 2, 4, 8, 16 min. Low dose rate irradiations (0.1 mGy/h ∼ 90 mGy/h) for 1 or 2 days were used by γ-simulator (137Cs γ-source: 1.1Tbq, 110Gbq) in IES. The irradiated murine NIH/PG13 cells were fixed by 4% parahormaldehyde immediately after irradiation, and reacted with 1st antibody (anti-γH2AX or anti-p53BP1). Alexa 488 or Alexa 647 conjugated Anti IgG were used for immunocytochemical analysis. The number and size of foci were used In Cell Analyzer (GE Healthcare).
§Results and Disucussions§ Numbers of γH2AX and p53BP1 foci were increased depending on total dose and dose rate in middle dose rate irradiations (more than 9 mGy/h). On the other hand, in low dose rate irradiations (less than 1.5 mGy/h), their focus formation were independent on total dose and dose rate, which was shown like reverse dose rate effect. These results indicate that DNA repair in low dose rate irradiations less than 1.5 mGy/h have a totally different molecular mechanism from that in middle and high dose rate irradiations. (This work was supported by Aomori Prefecture, Japan.)

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An adaptive response induced by low dose-rate long-term irradiation in mice were evaluated in terms of the amount of DNA damage. C57BL/6N female mice were irradiated with 0.5Gy of¹³⁷Cs-γ rays at 1.2mGy/hr. Then challenge dose (0.5, 1 or 2Gy) was given. For detection of initial DNA damage, we modified single cell gel electrophoresis technique (comet assay). A certain reduction in the amount of DNA damage was observed in pre-irradiated mice spleen cells compared to mice which received the challenge dose only; an adaptive response in terms of DNA damage was induced by low dose-rate long-term irradiation in mice.
One of the possibilities for the mechanism of reduction of initial DNA damage was an enhancement of antioxidative capacity which would protect DNA from injury by reactive oxygen species. Therefore, we measured the level of antioxidants in the spleen of mice irradiated with 0.5Gy at the low dose-rate; a significant increase in the gene expression of catalase was observed. These results suggest that induction of the antioxidants was involved in the adaptive response in terms of DNA damage in mice irradiated with the low dose-rate irradiation.

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We investigated that the effects of priming dose on the frequency of apoptosis and the expression of p53 in C57BL/6N mouse spleen. Mice received a whole body irradiation with 0.02Gy (667μGy/min, 0.8μGy/min) and 2Gy (0.91µGy/min) γ rays as follows; control: no treatment, group 1: 0.02Gy (667μGy/min), group 2: 0.02Gy (0.8μGy/min), group 3: 2Gy, group 4: 0.02Gy (667μGy/min) + 2Gy, group 5: 0.02Gy (0.8μGy/min) + 2Gy. The frequency of apoptosis and p53 expression were measured using TUNEL method and western blot. For the dose-rate effect study, the challenge dose was exposed when priming dose finished. There were no differences in the frequency of apoptosis among the control, group 1 and 2. The frequency of apoptosis in the group 3 and 4 significantly increased, and there was no difference between these groups. However, the frequency of apoptosis in the group 5 was approximately half of the group 3. For the time interval study, 0.02Gy (667μGy/min) was pre-irradiated. The frequencies of apoptosis were significantly lower in the primed mice at the time intervals of 2 to 168 h than those in the group 3. The expression of p53 in the group 2 increased compared that in the group 3. Furthermore, in group 4 and 5, the p53 expression increased compared to the group 3. In the group 5, the expression was maximum level. There were no expression of p53ser15 in the control, group 1 and 2. In group 3, its expression was high level, but low in group 4 and 5,We suggest that p53 stimulates repair system and then protects from apoptosis in the adaptive response.

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Low dose radiation is reported to have beneficial effects such as attenuation of diabetes, autoimmune diseases, and cancer, which is called radiation hormesis. Because the disorder of accommodation in immune system is involved in such diseases, immunological network is assumed one of the targets for radiation hormesis. We have shown that continuous irradiation with low dose-rate gamma-ray enhances cellular immunity. Because hyper-activation of humoral immunity is involved in autoimmune diseases such as rheumatoid arthritis, it is expected that enhancement of cellular immunity by low dose-rate irradiation results in suppression of enhanced humoral immunity and amelioration of these autoimmune diseases. Here, we evaluated efficacy of continuous irradiation with low dose-rate gamma-ray on mouse experimental model for collagen-induced arthritis. Joint inflammation of mice immunized with 0.05 mg collagen in Freund's complete adjuvant, was detected 7 days after the immunization. Maximum inflammation was observed 40-50 days after the immunization, and gradually reduced thereafter. Continuous irradiation with 0.01-1 mGy/h gamma-ray significantly retarded and reduced the inflammation. The irradiation reduced serum IgM and IgG specific for mouse collagen, and recovered ability of splenocytes to produce interferon-gamma, which was suppressed by the immunization. Our results suggest that continuous irradiation with low dose-rate gamma ray is effective on collagen-induced arthritis at least in part through the enhancement of cellular immunity.

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We have reported that low-dose irradiation enhanced the immune and antioxidation function and reduced the oxidative damage by the immuno-assay and bio-assay. In the present study we examined the histological changes of low-dose X-ray irradiation on spleen, ovary and liver in mouse. Female BALB/c mice, 7 weeks of age, were irradiated by sham, 0.25 Gy, 0.5 Gy, or 15 Gy (reference) of X-ray. Each mouse was killed at 4 hrs, 24 hrs, or 48 hrs after irradiation. Paraffin sections of those organs were stained with hematoxylin-eosin. In the results, the histological examinations of spleen sections revealed that at 4 hrs after 0.25Gy irradiation and at 24 and 48 hrs after 0.5Gy irradiation, lymphatic follicles (white pulp) were larger than those of sham irradiation, and that at 24 and 48 hrs after 15Gy irradiation, lymphatic follicles decreased sharply. In granulose cells and stromal cells of the ovary, apoptosis was induced at 4, 24 or 48 hrs after each irradiation. In the liver, there were no differences among each irradiation. These findings suggested that the lymphocytes in the spleen were increased by 0.25Gy or 0.5Gy irradiation, and such low dose irradiation enhances immune function. This study is required to evaluate the cytoarchitecture of T cells and B cells in spleen, thymus and bone marrow. We will also report the results.

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In this study, we investigated the effects of a low dose preirradiation on radiation-induced lethality in Caenorhabditis elegans. C.elegans were maintained at 20°C on solid nematode growth medium (NGM) with Escherichia Coli OP50 for food. Wild-type N2 Bristol strain was used in this experiment. As a preirradiation, synchronous L4 stage larvae or young adults were irradiated with 5 Gy γ-rays. After 18-24 hours incubation, animals were irradiated with lethal dose (75 Gy) γ-rays, and they were transferred to fresh NGM plates. Adults were removed 3 hours after 75 Gy γ-ray irradiation and then the subsequent survivals of laying eggs were counted for 3 days. In some case, the survival ratio in a preirradiated group was higher than that in a single irradiated group. Growth was strongly suppressed in all experimental groups.

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We simulated a hot spring condition using the mineral powder and measured the radioactivities and negative air ions in some conditions, to elucidate the various characteristics of the radioactivity and negative air ion originated from the Badgastein (Austria) radon hot spring. In the result, the radioactivity of the uranium series nuclides(²¹⁴Bi) in the mineral was 95 times of that of the thorium series nuclides(²⁰⁸Tl). For the pH (3-11) dependence of the leaching nuclides from the mineral in water, the uranium series nuclides (²¹⁴Bi, ²¹⁴Pb) and the thorium series nuclides (²¹²Pb) were well leached on the strong acid side than on the alkaline side. Moreover, there were many negative air ions originated from the mineral in the place within 10cm away from the mineral surface. This suggested that most negative ions exist within the range of the radiated α rays. Negative air ions increased with increasing atmospheric relative humidity (35-60%). This suggested that negative air ions stably existed in the hydration type such as O₂^-(H₂O)_n. Furthermore, for the hydrous rate dependence of the negative air ions originated from the mineral, the negative air ions without supernatant fluid were larger than those with supernatant fluid, because α rays ionized water on the surface of the mineral.

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Hot springs containing thoron (220Rn), which mainly emits a-rays, are thought to have curing effects on hypertention. We first examined the temporal changes in antioxidants and vasoactive substances in human blood to elucidate the mechanism of alleviation of hypertention by inhalation in the thoron hot spring (6130 Bq/L). Every 2 days, nasal inhalation of vapor from the hot spring in the room was performed for 40 min under a condition of high humidity (90 %). Blood samples were collected after each a bathing on 1 weeks, 2 weeks, and 3 weeks after first bathing, a blood sample also was collected before the first bathing to be used as the control. Results showed that thoron inhalation enhanced the antioxidation function (the increase of catalase activity), and the finding suggestes that thoron inhalation contributes to the prevention of hypertention related to peroxidation reactions. Moreover, the changes in vasoactive substances (dopamine and α-ANP) indicated increase in tissue perfusion brought about by thoron inhalation, suggesting that thoron inhalation plays a role in alleviating hypertention. The findings suggest that an appropriate amount of active oxygen is produced in the body after thoron inhalation, and this contributes to the alleviation of the symptoms of active oxygen diseases such as hypertention.

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On study of effects of X–rays on human body, an irradiation to experimental animals such as mice is indispensable. However, when X–ray energy is low (keV), depth doses are significantly attenuated by tissue absorption. In this study, the depth doses of mice are evaluated when added filters (aluminum, copper and tantalum) are placed in front of the X–ray tube. Preliminary study is X–ray attenuation by the Mix–DP phantom that was performed using the typical X–ray generator unit and the 0.03 cm³ parallel plate ionization chamber. The average axis depth of mice was estimated as about 2.5 cm; the Mix–DP phantom thickness was varied from 0.5 to 2.5 cm. Focal chamber distance (FCD) was 60 cm, and the exposure X–ray tube voltages were range of 75–150 kV. Added filters including heavy metal filter such as tantalum were used for X–ray beam hardening. On the other hand, ¹³⁷Cs γ–ray attenuation by Mix–DP was measured for comparison of X–rays. As a result, X–ray tube voltage in 150 kV, the filters that 0.5 mm aluminum, 0.2 mm copper, and 0.03 mm tantalum combined provide approximately the same effect as ¹³⁷Cs γ–ray, when the thickness of the irradiated material is equal to that of mouse. The combination of added filters resulted in the increasing tube load compared with no added filter (2.4 times of the exposure time). Moreover, the experimental result of which TLD chips are embedded in various mice will be reported.

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To elucidate the various characteristics of the radioactivity and negative air ion originated from an artificial thoron hot spring, we simulated a hot spring condition using a monazite powder and measured the radioactivities and negative air ions in the condition. In the result, the radioactivity of the thorium series nuclide (²⁰⁸Tl) in monazite was 5.3 times of that of the uranium series nuclide (²¹⁴Bi). For the pH (3.5-12.5) dependence of the leaching nuclides from monazite in water, the thorium series nuclides (²²⁸Ac, ²¹²Pb) were well leached on the strong acid side. On the other hand, the uranium series nuclides (²¹⁴Bi, ²¹⁴Pb) leached in water were not detected because the specific activities of the uranium series nuclides are lower relative to those of the thorium series nuclide. Moreover, there were many negative air ions originated from monazite in the place within 10 cm away from the monazite surface. This suggests that most negative ions exist within the range of the radiated &alpha rays. Negative air ions increased with increasing atmospheric relative humidity (35-60%). This suggests that negative air ions stably existed in the hydration type such as O₂^- (H₂O)_n. Furthermore, for the hydrous rate dependence of the negative air ions originated from monazite, the negative air ions without supernatant fluid were larger than those with supernatant fluid, because &alpha rays ionized water on the surface of monazite.

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Pre-irradiation with 0.5Gy of X–rays to C57BL/6N mice confers resistance to the bone marrow death caused by the following 6Gy-irradiation. We previously demonstrated that hyper-proliferation of bone marrow stem cells initiated by pre-irradiation to mice was not responsible for the acquisition of radiation resistance to bone marrow death, and speculated that the pre-irradiated bone marrow stem cells might acquire radiation resistance by a new biological function rather than hematopoietic function. In the present study, we prepared radiation resistant bone marrow cells from C57BL/6N mice primed by 0.5 Gy-irradiation, irradiated the cells in vitro, and then examined the spleen colony forming ability at 12th day after grafting the irradiated bone marrow cells into the recipient mice. The result indicated that the extent of radiation resistance was different in the bone marrow stem cells prepared at 8th day and 14th day after pre-irradiation. The bone marrow stem cells derived from the mouse at 14th day after pre-irradiation showed more ability to rescue the bone marrow death even though the number of spleen colonies was similar, suggesting that radiation resistant status of bone marrow stem cells was gradually maturing in donor mice after pre-irradiation. To know a role of radiation response in this process, the kinetics of expressions of p53 and p21 genes and proteins is under investigation.

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HiCEP is a method for gene expression profiling that is based on the AFLPmethod and doesn't require any sequence information for its analysis.Compared to pre-existing similar procedures its false positive rate is verylow, enabling us to assign almost all signals to specific transcripts.HiCEP uses competitive PCR technology and is extremely sensitive, detectingeven one copy / cell and less than 1.5-fold expression changes.Here we have attempted to minimize the starting material required for HiCEPanalysis. The standard protocol requires approximately 1.0 to 2.0micrograms of messenger RNA for one analysis. We have been able to reducethis to 10 nanograms of total RNA. This is equivalent to the amount inabout 1,000 mammalian cells and is [0.0001-0.001%] 1/1,000-10,000 of theamount required using the standard protocol. We observed that thesensitivity clearly depends on the amount of starting material and thatstarting material is particularly scarce for rarely-expressed transcripts.Currently we are getting results using only 100 cells, but the sensitivityat this level needs improvement.

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In Caenorhabditis elegans (C. elegans), the effect of hormesis that extends the life-span by exposure to oxygen radicals for a short time is confirmed. We have clarified that the daf-16 gene, which is homologous to FOXO transcription factor, participates in the hormesis using AGE mutants in C. elegans. In general, it is thought that hormesis is caused by activation of antioxidant systems dependent on pre-exposure to low dosis of radicals. To clarify the mechanism of inducement in hormesis, the change in concentration of intracellular superoxide when exposed to low dosis of radicals was measured by the MPEC method. Furthermore, to demonstrate the activation of antioxidant systems when hormesis was induced immediately, the mRNA expression of antioxidant genes was measured using RT-PCR. As a result, the concentration of intracellular superoxide anion was decreased significantly by exposure to low dosis of oxygen radical that causes the life-span extension in the age-1 mutant. Moreover, mRNA expression of the antioxidant genes such as SOD and catalase in age-1 mutant was increased significantly than daf-16 mutant. This indirectly shows that antioxidant systems were activated by the exposure to low dosis of oxygen radicals. Therefore, two possibilities that antioxidant systems in C. elegans was necessary to induce the life-span extension due to hormesis, and the DAF-16 transcription factor had acted in the upstream for this activation, were suggested.

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An exposure condition of tritium radiation from nuclear fusion reactor could be a long-term exposure at low dose rate. The biological effects of low dose (rate) radiation are not clear because none of suitable detection system has been established. Regarding to mutation induction by high LET radiation such as neutrons, the reversed dose rate effect has been reported when the dose rate is lower than a certain value. On the other hand, it is not clear whether this phenomenon could be seen in the case of tritium radiation. To examine the low dose rate effect of tritium radiation, we established a hypersensitive mutation detection system using hamster cells carrying a human X-chromosome. We have tested mutation induction by tritiated water at dose rate between 0.13 and 4.4 cGy/h. Although mutation frequency seems to be slightly increased at lower dose rate tritium radiation, it was not statistically significant. Our results suggest that the reversed dose rate effect may not be seen for mutation induction by tritium radiation.

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Whether or not dose rate effects is observed in low dose rate region less than 60 mGy- 600 mGy/h has not been well studied. C3H mice of 8 weeks age were chronically exposed to up to 700 days with 20 mGy/day (approximately 1mGy/h) and 500 days with 1mGy/day (approximately 0.05mGy/h). Chronological change of chromosome aberration rates in spleen cells were observed along with accumulated doses in these two low dose rates. Unexpectedly, unstable-type aberrations were increased in two phase manner within 0-2Gy and 4-14Gy in 20mGy/day exposure, and also they were slightly increased up to 0.5Gy in 1mGy/day exposure, although such an unstable type of aberrations has been considered to increase up to some dose range and they are saturated after that. Chromosome aberration rates in 20mGy/day and 1mGy/day were compared at the same total doses at 0.5Gy and 0.25Gy, which were shown as 2.0 vs. 0.53, and 1.0 vs. 0.47 respectively. In the calculation, chromosome aberration rates increased by age were subtracted from the observed aberration rates. Dose-rate effect was observed in the low dose rates region. An increase of unstable type of aberrations along with the accumulated doses may be caused from balance between cell growth and apoptosis, long lived lymphocytes or chromosomal instability. These results will be important to understand the mechanisms of low dose rate radiation –induced cancer development and risk assessment. The present study was supported by grant-in aid from Aomori Prefecture.

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Mice chronically irradiated with gamma-ray at a low dose rate (21mGy/day, 400 dyas) had significantly shorter life spans compared to the nonirradiated controls. The major cause for this life shortening was considered to be the earlier genesis and/or the faster progression of malignant lymphomas in the irradiated group. In the present study, chromosomal aberrations in 82 malignant lymphomas were analyzed by array-CGH method and were compared between the nonirradiated and the irradiated groups. Many array-CGH profiles showed numeric aberrations of whole-chromosome, of which trisomy 15 was most frequent in the irradiated group, as well as copy-number imbalances of partial chromosomal regions. Most of them are syntenic to human chromosomal regions frequently showing aberrations in human lymphomas, suggesting an involvement of common molecular pathogenesis. Gains on chromosomes 12 (p=0.005) and X (p=0.084) were associated preferentially with the nonirradiated group, while gain on chromosomes 11 (p=0.057) and losses on chromosomes 4 (p=0.088) and 14 (p=0.026) were detected more frequently in the irradiated group. Notably, recurrent aberrations detected in the irradiated group involved the following genes, such as c-myc , p16INK4a, p73, Rel and RB, where their dysfunctions or copy-number alterations were considered to confer increased aggressivity and proliferative advantage in human lymphomas. These findings suggest a possibility that highly aggressive lymphomas might develop preferentially in the irradiated group. This work was supported by grants from Aomori Prefecture, Japan.

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Micronucleus formation is used as a simple method to assess the biological effects of radiation instead of chromosomal aberration. Micornucleus in binuclear cells during the culture with cytochalasin B derived from lymphocytes in peripheral blood cells are usually observed. It is applied to a method of bioassay to assess radiation dose. We used it as one of the indices for the assessment of the effects of low dose/low dose rate radiation because the assessment needs a number of data for statistical use. The cell culture under irradiation continues growth since the cells are cultured for a long time without serious damage. There is possibly a difference in the micronucleus formation rate between the binuclear cells from growing cultured cells and those from lymphocytes derived from one step culture. There is because lymphocytes in peripheral blood originally may have no cells with micronuclei whereas some cells in the growing culture have them. It is alos possible that cells with micronucleus will divide further. The difference was assessed with a model based on recurrence relation under several simple hypotheses.

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It is important for radiation risk estimation to study the adaptive response triggered by low-dose ionizing radiation. In this study, we tried to detect the adaptive response as genetic change on the chromosome by using the LOH (Loss of heterozygosity) detection system, which we have already established in human lymphoblastoid cell TK6. The priming doses of X-rays were 2.5 , 5, or 10 cGy , the challenging X-ray dose was fixed at 2 Gy and the interval between two irradiations were changed from 1 to 12 hr. An approximately 30 % recovery from the lethal effect of 2 Gy exposure was observed with the 5 cGy pre-irradiaiton at 3hr before the challenging exposure, but this recovery rate was not statistically significant. While the 6 hr proceeded 5 cGy pre-irradiation decreased the mutation frequency in thymidine kinase (TK) gene locus to about half of non-pre-irradiation level. The LOH analyses of the obtained TK mutants provided an interesting finding as follows; homozygous LOH events in the restricted chromosome region, probably caused by the non-homologous end-joining repair of DNA double-strand break, were observed only after the pre-irradiation although these events rarely occurred. The mechanisms responsible for this particular type of LOH events are now under the study.

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To investigate the mechanisms of the suppression of incidence of methylcholanthrene(MC)-induced fibrosarcoma by low dose-rate irradiation, we examined the tumor cell rejection in irradiated and non-irradiated mice using TD50 (tumor dose 50) assay.
C57BL/6N(B6) mice were irradiated with 137-Cs gamma-rays at 1.2mGy/hr until total dose reached 150, 250, 800, 1200 mGy. Then, various number of tumor cells prepared from MC-induced fibrosarcoma were injected to estimate TD50 values. An increased TD50 value, by a factor of 5, was observed only in the 250mGy irradiated mice. When the irradiation was prolonged after the tumor cell injection, the mice received pre-injection dose of 800 or 1200mGy also shows elevated TD50 values.
On the other hand, the values in irradiated scid mice with 250mGy at 1.2Gy/hr don't increase compared to the value in non-irradiated scid mice. This result suggests that the immune system is involved in the enhancement of the tumor cell rejection by the low dose-rate irradiation.

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We tried to confirm the previous findings that growth of Paramecium tetraurelia and mouse leukemia cells was suppressed by shielding the natural background radiation and that the growth suppression of the cells was restored by compensatory radiation from a low dose gamma-ray source. We used a radiation-shielding box surrounded with iron (15cm) and paraffin (10cm) walls, which can reduce the gamma-ray and neutron doses to 1/50 and 1/4 of the background levels, respectively. We found no effect of the background radiation-shielding on the growth of P. tetraurelia cells for 10 days. We also found no effect of additional low dose gamma-ray irradiation on the growth of the cells.As there was no effect of background radiation-shielding on the growth for 10 days, we conducted long term shielding experiment. The cumulative fission number of the P. tetraurelia was reduced by shielding the background radiation during the whole life span of the cells. Now, we are conducting the further experiment to confirm the result.We conducted the same experiment using mouse leukemia L5178Y cells. We found that the growth of the mouse cells was suppressed by shielding the background radiation for 5-7 days. We also found that the growth suppression of the cells was restored by compensatory radiation from a low dose gamma-ray source in the shielding box. These results were not found in the M10 cells that are the radiation hypersensitive mutant of L5178 cells. We are investigating the mechanism of these phenomena by a molecular method.

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For the risk estimate of radon exposure, the biological effects for the tracheal epithelial cells were examined by in vitro exposure to radon. The dosimetric evaluation of the absorbed dose to basal cells of the bronchial epithelium per unit exposure gives 9 nGy/(h Bq/m³) for average indoor radon conditions (UNSCEAR 2000). Recent epidemiological report indicates that the risk of lung cancer increased by 8.4 % per 100 Bq/m³. When an equilibrium equivalent factor is assumed to be 0.4, the absorbed dose rate is 360 nGy/h. The experimental dose rate of alpha rays is several mGy/min as usual. Therefore, there is a great difference between environmental and experimental dose rate. Our exposure system produces environmental dose rate as nGy/h level. For radon exposure of cells, a ceramic radon source that was developed for radon experimental research was used. The radon gas was injected to the incubator through the filter. The absorbed dose was evaluated by using energy spectrum data of the irradiated alpha rays to the cells. The tracheal epithelial cells were isolated from Wister rat (WM/NIRS), and cultured by the air-liquid interface (ALI) method. This culture method is expected to be especially useful for simulation of exposure to radon. The biological effects were examined among several dose rate of alpha rays.

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Linear non-threshold (LNT) model is a basic theory for radioprotection, but the adaptability of this hypothesis to the risk at very low doses or at very low dose rates is not sufficiently investigated. In this study a method for quantitative risk assessment in epidemiological studies investigating dose rate effects was proposed. For describing quantitatively the association between risk and dose rates, we applied some statistical models to experimental data at low dose with statistically sufficient accuracy. Data on micronucleus formation and [³H] thymidine uptake in human cells were analyzed using the Median Effective Dose (MED) as a measure of the risk. In this experimental data, it was obvious that mathematically estimated MED increased with longer irradiation time and that the biological response depends on not only cumulative dose but also irradiation time. We should point out that there is a dose rate range over which risk depends on both dose and dose rate, and that risk depends primarily on dose rate at very low dose rate. Furthermore the proposed approach was applied to some epidemiological studies that have been already reported. It was suggested that the model can describe the relationship between the risk and dose rate under some hypotheses in epidemiological studies. One of the advantages of the model is that we can subsequently test predictions for other experimental or epidemiological data because the model can describe a wide dose-rate range extending from background to experimental level radiation.

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Low LET radiation generates toxic free radicals and the resultant free radicals cause injury to nuclear DNA that makes serious damages in living cells. Although endogenous mechanisms to diminish toxicity of the free radicals has been studied in the cell-free system, their functions in the living cells are not clarified. To reveal the initial damages and the protection mechanism, simultaneous measurements of the micronuclei formation and growth arrest in murine macrophage cell line RAW264.7 were performed. Since the glutathion is a major protector for radicals in living cells, we focused on the enzyme glutathion peroxidase (GPx) that catalyzes conversion of glutathion from oxidative to reduced form. A vector that continuously expresses GPx-4 with nuclear localization signal (NLS) peptide sequence at the N-terminal locus was stably introduced into the cells. The frequency of growth arrested cells by radiation was decreased in the most of the transformants. When the NLS was replaced with other signal peptides or removed in the GPx-4 expression vector, the diminishment of the growth arrest was not observed. In any of the transformants, radiation-induced micronucleus rate was not changed. These results show that GPx4 in nucleus leads to cure radiation damages that triggers growth arrest.

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CAPE is known as an antioxidant, and it is proposed that the induction of HO-1 by CAPE is mediated by a redox-regulated mechanism. Therefore in this study we examined whether the activation of HO-1 gene by CAPE is dependent upon redox mechanisms. RAW cells were incubated with (1) CAPE, (2) caffeic acid ethyl ester (CAEE), (3) chlorogenic acid or (4) curcumin for 4 hrs. and mRNA of HO-1 gene was measured by RT-PCR. As a result the mRNA was enhanced by the chemicals as (1) 36.5, (2) 20.1, (3) 1.3 and (4) 6.4 times respectively. Then to measure reductive activities each chemical was subjected to the second-harmonic alternative current voltammetry. The one-electron oxidation potentials of these phenolic compounds were 0.48 (CAPE), 0.49 (CAEE), 0.48 (chlorogenic acid) and 0.42 (curcumin). Thus curcumin exhibited the highest reductive activity, and the others were less potent and almost at the same level. However the increase of HO-1 mRNA by curcumin was less than CAPE in spite of its higher reductive capacity, and also chlorogenic acid exhibited little activation of HO-1 gene in spite of the same level of reductive capacity as CAPE. When we compare the structure of CAPE and chlorogenic acid, it is presumed that a big substituent of chlorogenic acid might interrupt the interaction with cellular components but not in CAPE. From these results we suggest that the transcriptional activation of HO-1 gene by CAPE and CAEE is not dependent on a redox-regulated mechanism, but on a structure-specific interaction mediated by receptors.

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One of the mechanisms of radiation-induced reoxygenation is a decrease in oxygen consumption of irradiated tumor tissues. In our previous study, we have reported that the cellular oxygen consumption rate in both L5178Y and M10 cells (a radiosensitive mutant of L5178Y) increased shortly after both X rays and 80 keV/µm carbon ions irradiation, and then returned to the value before irradiation. The time of occurrence of peak oxygen consumption for L5178Y and M10 was 24 hours after irradiation, and was independent of radiation quality and dose. In the present study, we measured the oxygen consumption rate in mouse lymphomas at 24 hours after X rays and carbon ions irradiation to investigate the dose dependency and the LET dependency of the cellular oxygen consumption rate. The peak oxygen consumption in both L5178Y and M10 at 24 hours after irradiation increased as dose increases to 20 Gy of X rays and carbon ions. The peak oxygen consumption in M10 was about 300% of controls, and was higher than that in L5178Y. The oxygen consumption in L5178Y at 24 hours after carbon ion irradiation increased as LET increases from 20 to 80 keV/µm.

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We have investigated bystander chromosomal damage in primary normal human bronchial epithelial (NHBE) cells irradiated with low-energy He-ion broad beams. Special 'strip' track segment dishes were made by cutting the Mylar surface on the bottom of special cell dishes into many equal strips and removing alternate strips. The remaining Mylar strips are sufficiently thick to stop the incident He ions, so that cells placed on these surfaces are never irradiated with He ions. When the unirradiated cells were kept with He-ion irradiated cells on the dishes, bystander chromosomal effect was observed in the unirradiated bystander cells. Many types of chromatid-type fragments, such as chromatid break, acentric fragment, isochromatid deletion and interchromatin exchange, were observed in the direct hit cells. On the other hand, only chromatid breaks and acentric fragments were observed in the bystander cells. There is evidence that simple chromosomal damage occur in bystander chromosomal effect.

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Purpose: We have evaluated the physiological functions that supervise radiation-sensitivity of human cells. The functions seem to be regulated by serum factors such as cytokines in the human body. This study examined the involvement of GRP78 in suppression of radiation-induced mutagenic events by interferons in human cells.
Methods: Human RSa cell line and its derivative variant cells, with and without interferons (α or β) treatment, were used for estimation of their susceptibility to UVC- or X-ray-induced phenotypic mutation (increased resistance to 6-thioguanine- or oubain-caused cell-killing) and that of DNA-repair capacity for excision of thymine dimers. Cellular contents of GRP78 were estimated by Western blotting analysis.
Results and discussion: GRP78 has been identified by the mRNA differential display method, as one of genes involved in enhancement of cell mutability by serum derived from pancreatic cancer patients. Variants, which expressed low levels of GRP78, showed higher frequency of phenotypic mutation and lower capacity of DNA excision repair than did the parent cells with normal expression of GRP78. In contrast, high celluar contents of GRP78 were found in interferon-treated cells. These results suggest that GRP78 may play some roles on interferon-induced radiation-resistance in human.

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Cellular response to ionizing radiation is complex and involves the participation of different class of genes such as DNA repair, cell cycle control, signal transduction apoptosis and oncogenesis. The liver is one of the target organs of radiation-induced cancers by internal exposures. In order to elucidate radiation-induced liver cancers including Thorotrast, we present new approach to investigate in vivo effects of internal exposure to α-particles. Adopting boron neutron capture, we separately irradiated Kupffer cells and endothelial cells in the mouse liver and the changes of gene transcriptions were analyzed by an oligonucleotide microarray. The number of differentially expressed genes was 166 out of 6,050 genes examined under criteria of up-regulation as 3-fold and under-regulation as less than 0.3-fold compared with un-irradiated mice. Real-time PCR validated the results of the microarray analysis. Genes with altered expression were different between exposures to α-particles and γ-rays. Gene expression profile revealed that the liver was in an inflammatory status characterized by up-regulation of positive acute phase protein genes irrespective of target cells of radiation exposure. In comparison with chemical and biological hepatotoxicants, inductions of Metallothionein1 and Hemopexin, and suppressions of G0s2 and cytochrome P 450s are characteristic to radiation exposure. Antiinflammatory treatment could be helpful for the prevention and protection of radiation-induced injury.

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We have improved the antisense method for the suppression of specific gene expression by introducing photo-induced binding of antisense oligonucleotides labeled with photo-reactive fluorescein (F-DNA) to a target sequence. Using F-DNA targeted to p53 gene, we have studied 1) in vitro photochemical binding of 18 mer F-DNA to the target 54 mer oligonucleotides including complementary sequence to F-DNA using 15 % polyacrylamide gel electrophoresis and 2) suppression of X-ray or UV-B induced p53 protein in human keratinocytes detected by Western blot. The formation of a complex with higher molecular weight was detected upon visible light irradiation in the in vitro system. Irradiation of F-DNA with no complementary sequence (scramble F-DNA) did not produce such a higher molecular weight band. Cellular experiments showed that irradiation of visible light to cells which incorporated F-DNA induced further reduction of p53 level in addition to the normal antisense effect, while use of scramble F-DNA exhibited little suppression. These results strongly suggest that the present "photo-antisense method" has a potential for biological and medical applications to regulate specific gene function.

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This study is intended to elucidate the molecular mechanism of the mutagenesis caused by ion beam. The mutation sites caused by ion beams were determined and the mutation spectrum were compared with those induced by gamma ray.
S. cerevisiae strains used in this study are S288C(RAD⁺), X36B-3C(rad3), JG-18(rad18), and G160/2b(rad52).
The yeast strains were irradiated with carbon ions (¹²C⁵⁺; 220 MeV) with the dose 10 to 300 Gy, and LET is 107 keV/µm. Carbon ion beam was generated from AVF cyclotron in JAERI. The mutation sites of ura3 mutants were determined by DNA sequencing.
The results show that the optimum dose for mutagenesis by carbon ion beams is 100 Gy. The ion beams at 100 Gy generate mutations 168.5 fold more than spontaneous mutation.
A portion of the URA3 regions (804bp) was sequenced. Mutations generated by ion beams are dominantly transversion, whereas, mutations of spontaneous generation are usually transition. The transversion / transition ratio in the WILD type was 5.0.
Moreover, the fact that GC → TA transversion were largely observed suggests that the mutations by ion beams resulted from oxidative damage such as formation of 8-oxodGTP mainly.
Further, the mutation sites are localized on several regions with 170bp to 186bp interval. This interval may correspond to nucleosome structure.

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We have found long-lived radicals (LLRs) in irradiated mammalian cells by direct measurement of ESR spectroscopy. LLRs are identified as sulfinyl (SLF radicals: R-CH₂-SO•) and hydrogen-added phenylalanine radicals (HPA radicals: R-CH₂-C₆H₆•), so these were generated in proteins. Levels of SLF radicals and frequency of hprt^– mutations in irradiated cells are simultaneously reduced when Vit. C is added AFTER irradiation, we proposed SLF radicals in proteins may induce DNA mutation. Investigation of formation mechanisms of LLRs is the first step to confirm the model. In this study, PBS suspensions of syrian golden hamster embryo cells saturated with nitrogen or oxygen gases were γ-irradiated at the room temperature followed by ESR measurements. When the nitrogen were saturated, HPA radicals were produced but SLF radicals not. In the case of oxygen, SLF radicals were produced well, and yield of HPA radicals have decreased to that in the nitrogen saturated suspensions. So SLF radicals are produced when solvated oxygen exist in the suspensions during irradiation. SLF radicals may be produced by the reaction cysteines with O₂^–. When the cells were saturated with N₂O as electron scavenger and irradiated, yield of HPA radicals decreased to almost 80% of that in irradiated nitrogen saturated suspensions. Thus HPA radicals are produced by hydrated electron addition to a phenyl ring followed by H⁺. LLRs are produced by indirect effect of radiation.

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The exposure of X-rays or heavy ion particles to diapause eggs of the black-striped strain (PS) silkworm, Bombyx mori, resulted in somatic mutations appearing as a white spot on the black integument during larval stage. This result indicates that the silkworm larvae from the eggs of the black-striped strain (PS) of the silkworm represent an appropriate model for estimating the biological effect of cosmic radiation, radiosensitivity of the eggs against X-rays and heavy ion particles (C and Ne beam) was examined as ground-based experiments. Ne beam irradiation of diapause eggs displayed dose- and linear energy transfer (LET)-dependent effects, causing a maximal rate of the mutation at 150 keV/㎛. Especially, a high incidence of somatic mutation (about 90%) was observed in the diapause-terminated eggs which received C, Ne, Fe beam on day 2 after the resumption of embryogenesis. In contrast, irradiation of Fe ion beam to the diapause terminated resulted in the occurrence of different type of somatic mutation, which showed the many tiny white spots on the black integument, from those in the same eggs irradiated with Ne beam. In the analysis of gene expression using RT-PCR-differential display method, a remarkable difference was observed in electrophoretic bands of amplified DNA derived from template RNAs of Ne beam irradiated eggs and non-irradiated eggs. This shows that the specific gene expression may be induced by irradiation of Ne beam to the diapause-terminated eggs.

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We have been investigating the molecular mechanism of biological effects caused by the low dose rate irradiation.We have focused on the mutagenic effects caused by various kind of irradiation at the low-dose rate by detecting the Hprt gene mutation that shows 6-thioguanine resistance using the mouse cultured cell line. Also the mutation spectrum in the 6-thioguanine resistant cells were analyzed by the PCR-based LOH analysis of the Hprt genome gene domain.In this session, the frequency of appearance and mutation spectrum in the Hprt deficient mutants obtained through the X-ray exposure at various dose rates will be presented.

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Micronucleus formation frequency of onion root tip cells induced by high LET radiations showed a characteristic dose-effect relationship; in the low dose region that averagely a few tenth of the charged particles traverse the cell nucleus, the frequency per dose is larger than that in higher dose region. It may be due to some specific phenomena such as by-stander effect in low dose rate low dose irradiations; we examined difference between high dose rate and low dose rate low dose irradiations intensively. But no significant difference could be found.
By examinations of the higher doses, we confirmed a bell-shaped dose-effect curve. The peak corresponds to dose that averagely from five to ten particles traverse the cell nucleus. A mathematical model was constructed using a: micronucleus formation frequency per particle traversal, n: number of the particles traversing the cell nucleus following a Poisson distribution of average m, μ: frequency of the micronucleus, and S_n: surviving probability of a cell of which nucleus is traversed by n particles escaping direct death or mitotic delay. Observed value could be explained with the model; the micronucleus formation was found to be strongly related to a character of radiations, which is assembly of particles.
We now investigate LET dependence of a and S for heavy ions, and dose-effect relationship of X-rays, to make comprehensive evaluation of the micronucleus formation.

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We examined mutations at the Hprt locus in 6-thioguanine-resistant mutants of mouse m5S cells arising spontaneously or induced by irradiation with X-rays. Unexpectedly, approximately one third of X-ray-induced mutants did not exhibit any DNA sequence alterations at the Hprt gene. Analysis with RT-PCR of the Hprt gene revealed that no trace of the Hprt cDNA was detected in these mutants. We also found that some X-ray-induced mutants that had either a base substitution or a small deletion contained the reduced amount of the Hprt mRNA. In contrast, spontaneous mutants had normal amount of the Hprt mRNA. These results suggest that ionizing radiation causes a reduction of gene expression, which could result in mutant phenotype. Epigenetic change might be an important factor for mutagenesis induced by ionizing radiation.

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Cleft lip and/or cleft palate (CLP) is one of the most common malformations in new born infants and seem to be caused by a combination of genetic and environmental factors. A particular mouse strain, CL/Fr mice, shows a high incidence of CLP, suggesting involvement of genetic factors, though genes relevant are not identified. p53 tumour suppressor gene could affect CLP development because p53 prevents malformations such as exencephaly probably by inducing apoptosis of damaged cells that result in developmental abnormalities. On the other hand, there are reports that γ-irradiation, an environmental factor, increases the incidence of CLP in their offspring. p53 is a well-known sensor and signaling protein of radiation-induced damages, however effect of p53 on radiation-induced CLP is not investigated. Thus, we produced the p53-deficient CL/Fr and examined CLP in mouse embryos. p53-deficient allele of p53(KO/+)BALB/c was introduced into CL/Fr by a speed congenic method. p53(KO/+)CL/Fr generated are then mated each other and pregnant females were sacrificed on 18.5 day of gestation. Embryos were p53-genotyoped and the incidence of spontaneous CLP was examined. The incidence was 20% in p53(+/+) and p53(KO/+) and 13% in p53(KO/KO), indicating no significant difference between the two different classes of p53 genotype. This suggests that p53 does not impact the incidence of spontaneous CLP. Then, to know effect of radiation, we are examining embryos that were exposed to γ-irradiation on 9.5 day of gestation. Results of incidence and radiation effect will be reported.

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